RESUMO
The regulated formation and resolution of R-loops is a natural process in physiological gene expression. Defects in R-loop metabolism can lead to DNA replication stress, which is associated with a variety of diseases and, ultimately, with cancer. The proteins PARP1, DIDO3, and DHX9 are important players in R-loop regulation. We previously described the interaction between DIDO3 and DHX9. Here, we show that, in mouse embryonic fibroblasts, the three proteins are physically linked and dependent on PARP1 activity. The C-terminal truncation of DIDO3 leads to the impairment of this interaction; concomitantly, the cells show increased replication stress and senescence. DIDO3 truncation also renders the cells partially resistant to in vitro oncogenic transformation, an effect that can be reversed by immortalization. We propose that PARP1, DIDO3, and DHX9 proteins form a ternary complex that regulates R-loop metabolism, preventing DNA replication stress and subsequent senescence.
Assuntos
Replicação do DNA , Fibroblastos , Poli(ADP-Ribose) Polimerase-1 , Animais , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/fisiologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/fisiologia , Senescência Celular/genética , Carcinogênese/genéticaRESUMO
Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.
Assuntos
Morte Celular/genética , Expressão Gênica/genética , Expressão Gênica/fisiologia , Proteínas com Domínio MARVEL/genética , Proteínas com Domínio MARVEL/farmacologia , Melanoma/genética , Melanoma/patologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteolipídeos/genética , Proteolipídeos/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Animais , Antineoplásicos , Calreticulina/genética , Calreticulina/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Proteínas com Domínio MARVEL/metabolismo , Proteínas com Domínio MARVEL/fisiologia , Camundongos , Necrose , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Peptídeos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteolipídeos/metabolismo , Proteolipídeos/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Traumatic brain injury (TBI), often induced by sports, car accidents, falls, or other daily occurrences, is a primary non-genetically related risk factor for the development of subsequent neurodegeneration and neuronal cell death. However, the molecular mechanisms underlying neurodegeneration, cell death, and neurobehavioral dysfunction following TBI remain unclear. Here, we found that poly(ADP-ribose) polymerase-1 (PARP-1) was hyperactivated following TBI and its inhibition reduced TBI-induced brain injury. Macrophage migration inhibitory factor (MIF), a newly identified nuclease involved in PARP-1-dependent cell death, was translocated from the cytosol to the nucleus in cortical neurons following TBI and promoted neuronal cell death in vivo. Genetic deletion of MIF protected neurons from TBI-induced dendritic spine loss, morphological complexity degeneration, and subsequent neuronal cell death in mice. Moreover, MIF knockout reduced the brain injury volume and improved long-term animal behavioral rehabilitation. These neuroprotective effects in MIF knockout mice were reversed by the expression of wild-type MIF but not nuclease-deficient MIF mutant. In contrast, genetic deletion of MIF did not alter TBI-induced neuroinflammation. These findings reveal that MIF mediates TBI-induced neurodegeneration, neuronal cell death and neurobehavioral dysfunction through its nuclease activity, but not its pro-inflammatory role. Targeting MIF's nuclease activity may offer a novel strategy to protect neurons from TBI.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Degeneração Neural/metabolismo , Poli(ADP-Ribose) Polimerase-1/fisiologia , Animais , Morte Celular , Masculino , Camundongos , Camundongos KnockoutRESUMO
Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in processes of cell cycle regulation, DNA repair, transcription, and replication. Hyperactivity of PARP-1 induced by changes in cell homeostasis promotes development of chronic pathological processes leading to cell death during various metabolic disorders, cardiovascular and neurodegenerative diseases. In contrast, tumor growth is accompanied by a moderate activation of PARP-1 that supports survival of tumor cells due to enhancement of DNA lesion repair and resistance to therapy by DNA damaging agents. That is why PARP inhibitors (PARPi) are promising agents for the therapy of tumor and metabolic diseases. A PARPi family is rapidly growing partly due to natural polyphenols discovered among plant secondary metabolites. This review describes mechanisms of PARP-1 participation in the development of various pathologies, analyzes multiple PARP-dependent pathways of cell degeneration and death, and discusses representative plant polyphenols, which can inhibit PARP-1 directly or suppress unwanted PARP-dependent cellular processes.
Assuntos
Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Reparo do DNA/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Polifenóis/metabolismo , Polifenóis/uso terapêuticoRESUMO
The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.
Assuntos
Comportamento Animal , Encéfalo , Dano ao DNA , Reparo do DNA , Neurônios , Poli(ADP-Ribose) Polimerase-1 , Sono , Proteínas de Peixe-Zebra , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: Poly(ADP-ribose) polymerase1 (PARP1) interacts and poly(ADP-ribosyl)ates telomere repeat binding factor 2 (TRF2), which acts as a platform to recruit a large number of proteins at the telomere. Since the discovery of TRF2-SLX4 interaction, SLX4 is becoming the key player in telomere length (TL) maintenance and repair by telomere sister chromatid exchange (T-SCE). Defective TL maintenance pathway results in a spectrum of diseases called telomeropathies like dyskeratosis congenita, aplastic anemia, fanconi anemia, cancer. We aimed to study the role of SLX4 and PARP1 on each other's telomere localization, T-SCE, and TL maintenance in human telomerase-negative osteosarcoma U2OS cells to understand some of the molecular mechanisms of telomere homeostasis. MATERIALS AND METHODS: We checked the role of SLX4 and PARP1 on each other's telomere localization by telomere immunofluorescence. We have cloned full-length wild-type and catalytically inactive mutant PARP1 to understand the role of poly(ADP-ribosyl)ation reaction by PARP1 in telomere length homeostasis. TL of U2OS cells was measured by Q-FISH. T-SCE was measured by Telomere-FISH. KEY FINDINGS: We observed that SLX4 has no role in the telomere localization of PARP1. However, reduced localization of SLX4 at undamaged and damaged telomere upon PARP1 depletion was reversed by overexpression of exogenous wild-type PARP1 but not by overexpression of catalytically inactive mutant PARP1. PARP1 depletion synergized SLX4 depletion-mediated reduction of T-SCE. Furthermore, SLX4 depletion elongated TL, and combined insufficiency of SLX4 with PARP1 further elongated TL. CONCLUSION: So, PARP1 controls SLX4 recruitment at telomere by poly(ADP-ribosyl)ation reaction, thereby regulating SLX4-mediated T-SCE and TL homeostasis.
Assuntos
Poli(ADP-Ribose) Polimerase-1/metabolismo , Recombinases/metabolismo , Troca de Cromátide Irmã/fisiologia , Linhagem Celular Tumoral , Cromátides/metabolismo , Cromátides/fisiologia , Reparo do DNA , Homeostase , Humanos , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Recombinases/genética , Recombinases/fisiologia , Telomerase/metabolismo , Telômero/fisiologia , Homeostase do Telômero/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.
Assuntos
Pancreatite/fisiopatologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Acinares/metabolismo , Doença Aguda , Animais , Ceruletídeo/farmacologia , Modelos Animais de Doenças , Fibrose , Inflamação/patologia , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/imunologia , Pancreatite Crônica/patologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bone is a dynamic organ maintained by tightly regulated mechanisms. With old age, bone homeostasis, which is maintained by an intricate balance between bone formation and bone resorption, undergoes deregulation. Oxidative stress-induced DNA damage, cellular apoptosis, and cellular senescence are all responsible for this tissue dysfunction and the imbalance in the bone homeostasis. These cellular mechanisms have become a target for therapeutics to treat age-related osteoporosis. Genetic mouse models have shown the importance of senescent cell clearance in alleviating age-related osteoporosis. Furthermore, we and others have shown that targeting cellular senescence pharmacologically was an effective tool to alleviate age- and radiation-induced osteoporosis. Senescent cells also have an altered secretome known as the senescence associated secretory phenotype (SASP), which may have autocrine, paracrine, or endocrine function. The current review discusses the current and potential pathways which lead to a senescence profile in an aged skeleton and how bone homeostasis is affected during age-related osteoporosis. The review has also discussed existing therapeutics for the treatment of osteoporosis and rationalizes for novel therapeutic options based on cellular senescence and the SASP as an underlying pathogenesis of an aging bone.
Assuntos
Envelhecimento/fisiologia , Osso e Ossos/fisiologia , Osteoporose/tratamento farmacológico , Osteoporose/etiologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Animais , Osso e Ossos/fisiopatologia , Senescência Celular , Dano ao DNA , Epigênese Genética , Instabilidade Genômica , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Osteoporose/fisiopatologia , Hormônio Paratireóideo/metabolismo , Telômero/fisiologiaRESUMO
The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P = 0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.
Assuntos
Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antígeno B7-H1/fisiologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Fator de Transcrição AP-1/fisiologiaRESUMO
UV irradiation can injure the epidermis, resulting in sunburn, inflammation, and cutaneous tissue disorders. Previous studies demonstrate that EGFR in keratinocytes can be activated by UVB and contributes to inflammation. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and plays an essential role in DNA repair under moderate stress. In this study, we set out to understand how PARP-1 regulates UVB irradiation-induced skin injury and interplays with EGFR to mediate the inflammation response. We found that PARP-1 deficiency exacerbated the UVB-induced inflammation, water loss, and back skin damage in mice. In human primary keratinocytes, UVB can activate PARP-1 and enhance DNA damage upon PARP-1 gene silencing. Moreover, PARP-1 silencing and PARP inhibitor olaparib can suppress UVB-induced COX-2 and MMP-1 expression, but enhance TNF-α and IL-8 expression. In addition, EGFR silencing or EGFR inhibition by gefitinib can decrease UVB-induced COX-2, TNF-α, and IL-8 expression, suggesting EGFR activation via paracrine action can mediate UVB-induced inflammation responses. Immunoblotting data revealed that PARP-1 inhibition decreases UVB-induced EGFR and p38 activation. Pharmacological inhibition of p38 also dramatically led to the attenuation of UVB-induced inflammatory gene expression. Of note, genetic ablation of PARP-1 or EGFR can attenuate UVB-induced ROS production, and antioxidant NAC can attenuate UVB-induced EGFR-p38 signaling axis and PARP-1 activation. These data suggest the regulatory loops among EGFR, PARP-1, and ROS upon UVB stress. PARP-1 not only serves DNA repair function but also orchestrates interactions to EGFR transactivation and ROS production, leading to p38 signaling for inflammatory gene expression in keratinocytes.
Assuntos
Receptores ErbB/fisiologia , Inflamação/etiologia , Queratinócitos/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos da radiação , Ativação Transcricional , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Células Cultivadas , Ciclo-Oxigenase 2/genética , Reparo do DNA , Receptores ErbB/genética , Humanos , Interleucina-8/genética , Camundongos , Transdução de Sinais/fisiologiaRESUMO
The cross-talk between apoptosis and autophagy influences anticancer drug sensitivity and cellular death in various cancer cell lines. However, the fundamental mechanisms behind this phenomenon are still unidentified. We demonstrated anti-cancerous role of cisplatin (CP) and morin hydrate (Mh) as an individual and/or in combination (CP-Mh) in hepatoma cells and tumor model. Exposure of CP resulted in the production of intracellular reactive oxygen species (ROS)-mediated cellular vacuolization, expansion of mitochondria membrane and activation of endoplasmic reticulum (ER)-stress. Consequently, Cyt c translocation led to the increase of Bax/Bcl-2 ratio, which simultaneously triggered caspase-mediated cellular apoptosis. In addition, CP-induced PARP-1 activation led to ADP-ribosylation of HMGB1, which consequently developed autophagy as evident by the LC3I/II ratio. Chemically-induced inhibition of autophagy marked by increased cell death signified a protective role of autophagy against CP treatment. CP-Mh abrogates the PARP-1 expression and significantly reduced HMGB1-cytoplasmic translocation with subsequent inhibition of the HMGB1-Beclin1 complex formation. In the absence of PARP-1, a reduced HMGB1 mediated autophagy was observed followed by induced caspase-dependent apoptosis. To confirm the role of PARP-1-HMGB1 signaling in autophagy, we used the PARP-1 inhibitor, 4-amino-1,8-naphthalimide (ANI), HMGB1 inhibitor, ethyl pyruvate (EP), autophagy inhibitors, 3-methyl adenine (3-MA) and bafilomycin (baf) and small interfering RNAs (siRNA) to target Atg5 in combination of CP and Mh. Exposure to these inhibitors enhanced the sensitivity of HepG2 cells to CP. Collectively, our findings indicate that CP-Mh in combination served as a prominent regulator of autophagy and significant inducer of apoptosis that maintains a homeostatic balance towards HepG2 cells and the subcutaneous tumor model.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Cisplatino/farmacologia , Flavonoides/farmacologia , Neoplasias Hepáticas/patologia , Animais , Autofagia/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cisplatino/administração & dosagem , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/administração & dosagem , Proteína HMGB1/fisiologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Poli(ADP-Ribose) Polimerase-1/fisiologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a challenging cancer with little change in five-year overall survival rate of 50-60% over the last two decades. Radiation with or without platinum-based drugs remains the standard of care despite limited benefit and high toxicity. HNSCCs often overexpress epidermal growth factor receptor (EGFR) and inhibition of EGFR signaling enhances radiation sensitivity by interfering with repair of radiation-induced DNA breaks. Poly (adenosine diphosphate-ribose) polymerase-1 (PARP1) also participates in DNA damage repair, but its inhibition provides benefit in cancers that lack DNA repair by homologous recombination (HR) such as BRCA-mutant breast cancer. HNSCCs in contrast are typically BRCA wild-type and proficient in HR repair, making it challenging to apply anti-PARP1 therapy in this model. A recently published study showed that a combination of EGFR and PARP1 inhibition induced more DNA damage and greater growth control than each single agent in HNSCC cells. This led us to hypothesize that a combination of EGFR and PARP1 inhibition would enhance the efficacy of radiation to a greater extent than each single agent, providing a rationale for paradigm-shifting combinatorial approaches to improve the standard of care in HNSCC. Here, we report a proof-of-concept study using Detroit562 HNSCC cells, which are proficient for DNA repair by both HR and non-homologous end joining (NHEJ) mechanisms. We tested the effect of adding cetuximab and/or olaparib (inhibitors of EGFR and PARP1, respectively) to radiation and compared it to that of cisplatin and radiation combination, which is the standard of care. Our results demonstrate that the combination of cetuximab and olaparib with radiation was superior to the combination of any single drug with radiation in terms of induction of unrepaired DNA damage, induction of senescence, apoptosis and clonogenic death, and tumor growth control in mouse xenografts. Combined with our recently published phase I safety data on cetuximab/olaparib/radiation triple combination, the data reported here demonstrate a potential for combining biologically-based therapies that might optimize radiosensitization in HNSCC.
Assuntos
Cetuximab/farmacologia , Quimioterapia Adjuvante , Reparo do DNA/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/radioterapia , Proteínas de Neoplasias/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Cetuximab/administração & dosagem , Cetuximab/uso terapêutico , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Células Clonais , Terapia Combinada , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Ftalazinas/administração & dosagem , Ftalazinas/uso terapêutico , Piperazinas/administração & dosagem , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Estudo de Prova de Conceito , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5'-3'-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.
Assuntos
Exorribonucleases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Estruturas R-Loop/fisiologia , Células A549 , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Exorribonucleases/fisiologia , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Mutações Sintéticas Letais/genéticaRESUMO
In the last three months, the whole scientific community has shifted its focus to the fight against the COVI-2 infection (COVID-19) trying to use different medications to save the patients' life. In some studies, the results were completely inconclusive, as in the case of chloroquine. However, the recent discovery on benefits deriving from use of such anticoagulants for Covid-19 patients, has increased the success of patients' treatment. Among lots of old and new drugs, PARP-inhibitors were not considered as possible option in the treatment of Covi-2 infection, being the latter able to induce the inflammatory and thrombotic cascades. Since PARP-inhibitors are able to reduce and block mechanisms leading to thrombosis and inflammation, they could be used as antithrombotic medications. Therefore, the present brief report is aimed to open the discussion on the potentials of PARP-inhibitors in non-oncological settings, like Covid-19.
Assuntos
Anti-Inflamatórios/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/sangue , Fibrinolíticos/uso terapêutico , Pandemias , Pneumonia Viral/sangue , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Trombofilia/tratamento farmacológico , Trombose/prevenção & controle , Anti-Inflamatórios/farmacologia , COVID-19 , Infecções por Coronavirus/complicações , Reposicionamento de Medicamentos , Fibrinolíticos/farmacologia , Humanos , Inflamação , Pneumonia Viral/complicações , Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/fisiologia , SARS-CoV-2 , Trombofilia/etiologiaRESUMO
Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a 'PAR code'.
Assuntos
Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose , Animais , Fenômenos Fisiológicos Celulares , Células HeLa , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/química , Poli Adenosina Difosfato Ribose/fisiologiaRESUMO
This study aimed to explore whether PARP-1 regulatory pathway mediated X irradiation induced cell cycle arrest and apoptosis or not. In this regard, colonic mucosal injury caused by whole-body X-irradiation induced apoptosis through PARP-1, caspase 3 and p53 regulatory pathway were evaluated in experimental rat models. Eighteen Wistar albino rats were divided into three groups. Two radiation groups received 8.3â¯Gy dose of whole-body X-irradiation as a single dose and the control group received physiological saline intraperitoneally. Radiation groups were sacrificed after 6â¯h and 4 days of irradiation. PARP-1 and caspase 3 expression in the nuclei of colonic crypt cells significantly increased 6â¯h after irradiation, and declined 4 days after irradiation. In conflict with other studies that reported p53 as not being expressed widely in colonic mucosa, in our study the expressions of p53 were elevated both in the cytoplasm and in the nucleus of the crypt cells, especially 6â¯h after irradiation. In the radiation groups, colonic mucosal injury score was significantly elevated compared with that of the control group. Our data demonstrated that PARP-1, caspase-3 and p53 expression increased in colonic mucosa 6â¯h after irradiation.
Assuntos
Apoptose/efeitos da radiação , Colo/efeitos da radiação , Mucosa Intestinal/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Caspase 3/fisiologia , Colo/patologia , Feminino , Mucosa Intestinal/patologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/fisiologia , Raios XRESUMO
Rationale: Glioma is the most common primary malignant brain tumor in adults. Chemoresistance of temozolomide (TMZ), the first-line chemotherapeutic agent, is a major issue in the management of patients with glioma. Alterations of alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene constitute one of the most prevalent genetic abnormalities in gliomas. Therefore, elucidation of the role of ATRX contributing to TMZ resistance in glioma is urgently needed. Methods: We performed the bioinformatics analysis of gene expression, and DNA methylation profiling, as well as RNA and ChIP-seq data sets. CRISPR-Cas9 gene editing system was used to achieve the ATRX knockout in TMZ resistant cells. In vitro and in vivo experiments were carried out to investigate the role of ATRX contributing to TMZ resistance in glioma. Results: We found that ATRX expression was upregulated via DNA demethylation mediated by STAT5b/TET2 complex and strengthened DNA damage repair by stabilizing PARP1 protein in TMZ resistant cells. ATRX elicited PARP1 stabilization by the down-regulating of FADD expression via the H3K27me3 enrichment, which was dependent on ATRX/EZH2 complex in TMZ resistant cells. Magnetic resonance imaging (MRI) revealed that the PARP inhibitor together with TMZ inhibited glioma growth in ATRX wild type TMZ resistant intracranial xenograft models. Conclusions: The present study further illustrated the novel mechanism of the ATRX/PARP1 axis contributing to TMZ resistance. Our results provided substantial new evidence that PARP inhibitor might be a potential adjuvant agent in overcoming ATRX mediated TMZ resistance in glioma.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Proteína de Domínio de Morte Associada a Fas/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Glioma/tratamento farmacológico , Proteínas de Neoplasias/fisiologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Temozolomida/farmacologia , Proteína Nuclear Ligada ao X/fisiologia , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Sistemas CRISPR-Cas , Dano ao DNA , Reparo do DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Edição de Genes , Técnicas de Inativação de Genes , Glioma/genética , Glioma/metabolismo , Código das Histonas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Fator de Transcrição STAT5/fisiologia , Temozolomida/uso terapêutico , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Proteína Nuclear Ligada ao X/antagonistas & inibidores , Proteína Nuclear Ligada ao X/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Excessive light exposure is a detrimental environmental factor that plays a critical role in the pathogenesis of retinal degeneration. However, the mechanism of light-induced death of retina/photoreceptor cells remains unclear. The mammalian/mechanistic target of rapamycin (mTOR) and Poly (ADP-ribose) polymerase-1 (PARP-1) have become the primary targets for treating many neurodegenerative disorders. The aim of this study was to elucidate the mechanisms underlying light-induced photoreceptor cell death and whether the neuroprotective effects of mTOR and PARP-1 inhibition against death are mediated through apoptosis-inducing factor (AIF). METHODS: Propidium iodide (PI)/Hoechst staining, lentiviral-mediated short hairpin RNA (shRNA), Western blot analysis, cellular fraction separation, plasmid transient transfection, laser confocal microscopy, a mice model, electroretinography (ERG), and hematoxylin-eosin (H & E) staining were employed to explore the mechanisms by which rapamycin/3-Aminobenzamide (3AB) exert neuroprotective effects of mTOR/PARP-1 inhibition in light-injured retinas. RESULTS: A parthanatos-like death mechanism was evaluated in light-injured 661 W cells that are an immortalized photoreceptor-like cell line that exhibit cellular and biochemical feature characteristics of cone photoreceptor cells. The death process featured over-activation of PARP-1 and AIF nuclear translocation. Either PARP-1 or AIF knockdown played a significantly protective role for light-damaged photoreceptors. More importantly, crosstalk was observed between mTOR and PARP-1 signaling and mTOR could have regulated parthanatos via the intermediate factor sirtuin 1 (SIRT1). The parthanatos-like injury was also verified in vivo, wherein either PARP-1 or mTOR inhibition provided significant neuroprotection against light-induced injury, which is evinced by both structural and functional retinal analysis. Overall, these results elucidate the mTOR-regulated parthanatos death mechanism in light-injured photoreceptors/retinas and may facilitate the development of novel neuroprotective therapies for retinal degeneration diseases. CONCLUSIONS: Our results demonstrate that inhibition of the mTOR/PARP-1 axis exerts protective effects on photoreceptors against visible-light-induced parthanatos. These protective effects are conducted by regulating the downstream factors of AIF, while mTOR possibly interacts with PARP-1 via SIRT1 to regulate parthanatos. Video Abstract Schematic diagram of mTOR interacting with PARP-1 to regulate visible light-induced parthanatos. Increased ROS caused by light exposure penetrates the nuclear membrane and causes nuclear DNA strand breaks. PARP-1 detects DNA breaks and synthesizes PAR polymers to initiate the DNA repair system that consumes a large amount of cellular NAD+. Over-production of PAR polymers prompts the release of AIF from the mitochondria and translocation to the nucleus, which leads to parthanatos. Activated mTOR may interact with PARP-1 via SIRT1 to regulate visible light-induced parthanatos.
Assuntos
Luz/efeitos adversos , Parthanatos , Células Fotorreceptoras de Vertebrados , Poli(ADP-Ribose) Polimerase-1/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Fator de Indução de Apoptose/metabolismo , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
Poly(ADP-ribose) polymerase (Parp)-1 catalyzes polyADP-ribosylation using NAD+ and is involved in the DNA damage response, genome stability, and transcription. In this study, we demonstrated that aged Parp-1-/- mouse incisors showed more frequent dental dysplasia in both ICR/129Sv mixed background and C57BL/6 strain compared to aged Parp-1+/+ incisors, suggesting that Parp-1 deficiency could be involved in development of dental dysplasia at an advanced age. Computed tomography images confirmed that dental dysplasia was observed at significantly higher incidences in Parp-1-/- mice. The relative calcification levels of Parp-1-/- incisors were higher in both enamel and dentin (p < 0.05). Immunohistochemical analysis revealed (1) Parp-1 positivity in ameloblasts and odontoblasts in Parp-1+/+ incisor, (2) weaker dentin sialoprotein positivity in dentin of Parp-1-/- incisor, and (3) bone sialoprotein positivity in dentin of Parp-1-/- incisor, suggesting ectopic osteogenic formation in dentin of Parp-1-/- incisor. These results indicate that Parp-1 deficiency promotes odontogenic failure in incisors at an advanced age. Parp-1 deficiency did not affect dentinogenesis during the development of mice, suggesting that Parp-1 is not essential in dentinogenesis during development but is possibly involved in the regulation of continuous dentinogenesis in the incisors at an advanced age.
Assuntos
Polpa Dentária/patologia , Displasia da Dentina/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Incisivo/patologia , Odontoblastos/patologia , Odontogênese , Poli(ADP-Ribose) Polimerase-1/fisiologia , Fatores Etários , Animais , Polpa Dentária/metabolismo , Displasia da Dentina/patologia , Feminino , Incisivo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Odontoblastos/metabolismoRESUMO
The activation and recruitment of NK cells to the site of viral infection are crucial for virus control. However, it remains largely unknown what controls the recruitment of the activated NK cells to the infection site. In a model of intraperitoneal infection with vaccinia virus (VV), we showed that poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, is critical for NK cell recruitment to the site of infection and viral control in vivo. We further demonstrated that PARP-1 promotes the production of CCL2 and that the CCL2-CCR2 axis is essential for NK cell recruitment to the infection site. In addition, we demonstrated that peritoneal macrophages are the main producer of PARP-1-dependent CCL2 secretion. Mechanistically, PARP-1 functions as a regulator of NF-κB by promoting its nuclear translocation and binding to its response sequences in macrophages upon VV infection. Taken together, our results reveal a potentially previously unknown role for PARP-1-dependent CCL2 production in NK cell migration and viral control and may provide important insights into the design of effective NK cell-based therapies for viral infections and cancer.